Elucidating the Mechanisms of SA-4-1BBL Immunoprevention Through Advanced Informatics Approaches

Cancer immunoprevention leverages the immune system’s surveillance mechanisms to mitigate tumor development. Vaccines constituting a tumor antigen and an immune adjuvant are perceived immunoprevention modalities. However, relevant tumor antigens are unknown for non-viral cancers, which constitute the majority of human cancers. Our group has recently shown that SA-4-1BBL, a novel oligomeric recombinant agonist of CD137 costimulatory molecule, as a single agent, has robust immunoprevention efficacy against various tumor types. In marked contrast, agonistic antibodies to CD137 lacked such function. This study employs advanced informatics approaches for analyzing bulk RNA sequencing (RNA-seq) transcriptomics data to elucidate the molecular mechanisms driving SA-4-1BBL’s immunoprevention efficacy. Mice were treated subcutaneously with SA-4-1BBL or an agonistic antibody and injection site (IS) tissue and draining lymph nodes (LN) were analyzed for differentially expressed genes (DEGs). Our findings reveal that SA-4-1BBL induces a compartmentalized and temporally dynamic immune program, characterized by early effector activation at IS and sustained immune regulation in draining LN. K-means clustering of 4,564 DEGs identified 8 functionally distinct clusters, with IS-enriched clusters (1 and 4) upregulating effector genes such as Cd28, Klra1, Cd4, Cd40, and Cd40l, supporting CD4+ T and NK cell activation. In contrast, LN-dominant clusters (2 and 5) were enriched for regulatory genes (Tnfaip3, Irf5, Col1a2), implicating roles in tissue remodeling and long-term immune memory. Compared to agonistic antibody, SA-4-1BBL elicited a more selective and durable activation of adaptive immune pathways, including TCR signaling, Th1/Th2 differentiation, and NK cytotoxicity, while avoiding systemic inflammation. Agonistic antibody, in contrast, activated broader innate inflammatory programs, including Toll-like receptor and neurodegeneration-linked pathways. IMPRes analysis revealed that SA-4-1BBL engages sequential immune-regulatory circuits centered on Stat1, Cd247, and Ifng, and modulates the CD151–TGF-β axis, offering novel mechanistic insight into its immune-balancing effects. These findings define a compartmentalized immune architecture that underlies SA-4-1BBL’s ability to provide safe, long-lasting cancer immunoprevention. Our study highlights the value of applying comprehensive informatics techniques in decoding the spatial and functional complexity of tissue-specific immune programming.